Otitis media (OM) is the most common childhood bacterial infection and the leading cause of conductive hearing loss in children. Inflammation is a hallmark of OM. Although appropriate inflammatory response triggered by bacteria is essential for eradicating bacterial pathogen, excessive inflammatory response is clearly detrimental to the host due to severe tissue damage. To avoid overactive and detrimental inflammatory response, inflammation must be tightly regulated. Bacteria-induced negative feedback regulation is thought to play a critical role in preventing overactive inflammatory response by inhibiting Toll- like receptor-dependent signaling adaptors. However, the molecular mechanisms underlying the negative feedback regulation of inflammation in the pathogenesis of OM remain unknown. Our Long-term Objective is to understand the molecular mechanisms by which inflammation is induced and regulated in the pathogenesis of OM. During the previous grant period, we focused on investigating the key positive signaling pathways involved in induction of NF-kB-dependent inflammatory response by OM bacterial pathogens including NTHi and S. pneumoniae. Recently we found that CYLD, a newly identified novel deubiquitinase, acts as a negative regulator for NF-kB-dependent inflammatory response induced by NTHi in an autoregulatory feedback manner. Interestingly, CYLD is expressed at low level in middle ear under physiological conditions, but is greatly up-regulated by NTHi. These encouraging preliminary results have thus laid a solid foundation for us to fully investigate the negative feedback regulatory mechanisms by which NTHi-induced NF-kB-dependent inflammation is tightly controlled by CYLD in the pathogenesis of OM in vitro and in vivo (Hypothesis & Short-term Objective). Aim 1. Determine the key receptor-dependent signaling adaptors required for mediating NTHi-induced NF-kB-dependent inflammatory response. Aim 2. Determine the molecular mechanism by which CYLD inhibits NTHi-induced NF-kB-dependent inflammatory response via negative cross-talk with the TRAF6/7 adaptor complex. Aim 3. Determine the molecular mechanisms by which CYLD is induced and regulated by NTHi. Overall, the proposed studies will provide novel insights into the molecular mechanism underlying the tight regulation of inflammation in OM and may lead to new therapeutic strategies for OM patients. [unreadable] [unreadable] [unreadable]